The obesity pandemic brings with it multiple attendant metabolic comorbidities, including Type 2 Diabetes (T2D) and Non-Alcoholic Fatty Liver Disease (NAFLD). Both T2D and NAFLD are inadequately treated with currently available therapy; although multiple medications are approved for T2D, few address the underlying problem-insulin resistance. In addition, no medications are approved for NAFLD, the leading cause of chronic liver disease and fastest-growing reason for liver transplantation. Clearly, a wider net for potential therapeutics must be cast in order to stem the tide of obesity-related illness. Notch is a highly conserved family of proteins critical for cell fate decision-making, but less is known about Notch action in mature tissue. We showed that Notch signaling is present at low levels in normal physiologic conditions, but increases markedly in livers from diet-induced or genetic mouse models of obesity, and similarly in obese patients with T2D or NAFLD. As Notch has been shown to interact with FoxO1, the key transcriptional regulator of hepatic gluconeogenesis, and mTORC1, which regulates insulin-mediated lipogenesis, we hypothesized that Notch plays an active role in physiologic and pathologic glucose and lipid metabolism. We generated mice lacking hepatocyte Notch signaling- these mice, when challenged with high- fat diet feeding, showed improved glucose tolerance and a parallel decrease in hepatic steatosis. In proof-of- principle studies, we found that Notch inhibitor treatment of obese mice recapitulated our genetic loss-of- function model, suggesting Notch may be both a mechanistic node in obesity-related pathology as well as a bona fide therapeutic target. In this application, we will examine the mechanisms underlying activation of hepatic Notch signaling and its potential as a novel therapeutic target for obesity-induced metabolic complications. In Aim 1, we will determine how hepatocyte Notch signaling integrates with the insulin/FoxO1 and nutrient/mTORC1 pathways to modulate hepatic insulin sensitivity and triglyceride levels. In Aim 2, we study the mechanism by which Notch stabilizes and activates mTORC1. In Aim 3, we will study how the Notch signal is transduced - i.e., by what ligand, expressed by which cell type in the liver - and determine whether we can exploit this knowledge to design safe and specific Notch inhibitors for treatment of metabolic disease. Successful completion of this application will identify the underlying mechanism of inappropriate Notch signaling in obesity, as well as potentially repurpose existing Notch inhibitors for treatment of insulin resistance/T2D and NAFLD.